The huge worldwide competition in the biotechnology process industry puts forth several challenges. One of them is related to the ever-growing demands of governmental agencies that control environmental impacts. Thus, the search for new biochemical processes that are sustainable and, at the same time, economically competitive is a major driving force for this industry.
In this context, the use of enzymes in industry is increasingly intense. Enzymatic catalysis presents advantages when compared to classical processes:                (i) reactions occur at room temperature and pressure;        (ii) a specific reaction (reaction specificity) takes place on a particular site (site specificity). Therefore, the desired product may be obtained with high yields;        (iii) since substrate specificity is high, only the desired compound is selectively reacted, even if there are other species present in the same environment.        
However, enzymatic biocatalysts present drawbacks, namely:                (i) if free enzyme(s) is(are) employed, its(their) re-use in successive batches, or their recycle in continuous reactors, is not economical, because they are soluble in water and present in low concentration;        (ii) on the other side, if the enzyme(s) is (are) immobilized on insoluble supports, making possible the use in long journeys (repeated batches or continuous processes), the support matrices have, generally speaking, radii of the order of microns, to reduce pore diffusion effects. These biocatalysts (here understood as the support-enzyme assemble) have, in many instances, low shear stress resistance;        (iii) such low-dimension biocatalysts are difficult to separate from precipitated products, whenever they are present in the process.        
Additionally, enzyme activity may be reduced, sometimes irreversibly, by heat, by organic solvents, by acids or basis, or even by the reaction products. The proper biocatalyst (enzyme-support) engineering, however, may provide important gains in the catalyst life.
Research on immobilized biocatalysts has been performed since middle-50's. Presently, the following methods may be used for enzyme immobilization: (i) covalent binding, adsorption or ionic linking to the support; (ii) cross-linking, when enzyme molecules are interlinked by poly-functional reactants, and become insoluble; (iii) encapsulation, for instance in polymeric gel, microcapsules or lipid liquid membranes and (d) combinations of the previous methods.
Among these methods, gel encapsulation is more common, applied in the immobilization of several biocatalysts.
U.S. Pat. No. 4,975,375 describes a method for preparing a biocatalyst consisting of the following steps: (i) reducing the temperature of a reticulated polymeric gel, which has a specific phase transition temperature, and is able to reversibly expand or contract, expanding at temperatures lower than its phase transition's; (ii) adding the gel in the referred expanded state to a liquid medium with the enzyme, allowing the enzyme to migrate into it and (iii) heating at least a portion of the referred gel up to or above its transition temperature, what will cause its shrinking and, consequently, the entrapping of the enzyme.
Nevertheless, it should be pointed out that the classical methods have drawbacks, such as: (i) reducing enzyme activity after immobilization; (ii) lack of stability during the immobilization, making at least part of the molecules denature or change their conformation still outside the support; (iii) difficult reactivation of the catalyst; (iv) additional complications related to the immobilization reactions, which may be difficult to control or demand toxic reactants, hard to handle and (v) difficulties for the correct spatial orientation and precise positioning of the enzyme on the sites of interest of the support.
The association of a new method for enzyme immobilization with new configurations of integrate bioreactors is essential to make enzymatic processes feasible, complying with the reduction of environmental impacts while, at the same time, allowing a high process performance.
Bioreactors should preferably integrate, in the same equipment, the desired biochemical reaction, and stages of separation and purification of the desired product, such as its crystallization.
In the case of the production of semi-synthetic β-lactam antibiotics, one of the greatest drawbacks of the enzymatic route is its low selectivity, due to the competitive hydrolysis of the precursors (esters or amides) that would react with the β-lactam nuclei, and to the hydrolysis of the antibiotic itself. The same enzyme that promotes the synthesis catalyzes both undesired reactions.
The first semi-synthetic β-lactam antibiotic, derived after the substitution of the side-chain of penicillin G, was ampicillin (patented by Beecham, in 1961), followed by amoxicillin (Beecham patent, 1972) and by the semi-synthetic cephalexins (cephalexin, Lilly, 1970, cefazolin, Fujisawa, 1974, cephadroxil, BMS, 1977, among others). The evolution of the world market for these drugs was impressive ever since, from 1,000 ton/year in 1970 to 45,000 ton/year in 2000 (Moody H, Hogenboom A, Lange B, Heemskerk D, Dooren T V, Boesten W, Roos E. Enzymatic Production of Cephadroxyl. Oral communication and Abstract Book of the 10th European Congress on Biotechnology, CAT 12, Madrid, Spain, 8-11 Jul. 2001). These processes use chemical routes for the synthesis of the side-chains and for the condensation of the antibiotic, linking those chains to the proper β-lactam nucli (6-APA, nowadays obtained enzymatically from penicillin G, 7-ACA from cephalosporin C, and 7-ADCA, produced after the expansion, also via chemical route, of the thiazolidine ring of 6-APA). All these conventional processes are consolidated, but research is intense in this field, both in industry and academia, to establish an efficient enzymatic route.
To enhance the reaction selectivity, integrated reactors should be applied, with a biocatalyst constituted by the immobilized enzyme. In these reactors, antibiotic concentrations outrange their solubility limits in the process conditions. In this way, the desired product (in this case, the antibiotic) will precipitate before being attacked by the enzyme.
In conventional reactors, this process presents a series of drawbacks. In a well-mixed reactor, stirring will be too aggressive for the fragile particles of biocatalyst. Fluidized-bed reactors will demand high recycle flows, to sustain fluidization. Fixed-bed reactors may have serious mass transfer restrictions in the extra-particle film, decreasing the apparent reaction rates and, therefore, process productivity.
It is necessary, thus, to develop processes to manufacture immobilized enzymes, associated with integrated bioreactors that overcome the mentioned difficulties.